The polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterization and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers. The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Taq DNA polymerase thus serves to increase the specificity and simplicity of PCR.
In many PCR based reactions, a signal producing system is employed, e.g. to detect the production of amplified product. One type of signal producing system that is used in PCR based reactions is the fluorescence energy transfer (FRET) system, in which a nucleic acid detector includes fluorescence donor and acceptor groups. FRET label systems include a number of advantages over other labelling systems, including the ability to perform homogeneous assays in which a separation step of bound vs. unbound labelled nucleic acid detector is not required. A primary problem with many prior art techniques is linked to the synthesis of dual labelled fluorescent oligonucleotides. European Patent Application EP1726664 discloses a detection system which overcomes this problem by using single-labelled oligonucleotide sequences of differing melting temperature (Tm) that hybridise to one another in free solution to form a fluorescent quenched pair, that upon introduction of a complementary sequence to one or both sequences generates a measurable signal, one of the sequences being of a Tm that is below the annealing temperature (Ta) of the PCR process.
In detection systems using a labelled nucleic acid detector, high fidelity amplification is critical. Due to the nature of the PCR process and Taq DNA polymerase such methods can suffer from alternative side-reactions to the desired polymerisation reaction. For example, PCR can suffer from non-specific amplification when the reaction is assembled at ambient temperature. At sub-PCR temperatures, Taq polymerase retains a fraction of its activity and can therefore extend primers that are not complementarily annealed, leading to the formation of undesired products. The newly-synthesized region then acts as a template for further primer extension and synthesis of undesired amplification products. However, if the reaction is heated to temperatures of around 50° C. or above before polymerization begins, the stringency of primer annealing is increased, and synthesis of undesired PCR products is avoided or reduced.
Primer-dimer is also a common side-reaction affecting PCR. Accumulation of primer-dimer occurs because of the hybridisation and extension of the primers to each other. Formation of primer-dimer results in the depletion of the reagents and hence overall reduction of PCR efficiency.
Hot-start PCR is a method to reduce non-specific amplification and hence limit the formation of primer-dimers and many different approaches have been developed to achieve this see, for example, Moretti, T. et al. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. BioTechniques 25, 716-22 (1998) and Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance Nucleic Acids Res (2008) 36(20): e131. However, such techniques only achieve partial alleviation of such problems. As any error in sequences, non-polymerisation based reactions or primer mispriming such as primer dimerisation may cause the production of weak signal or the wrong signal being produced, particularly in allele specific PCR, further improvement of these weak or incorrect signals would be desirable.
Phosphorothioates (or S-oligos) are a variant of normal DNA in which one of the nonbridging oxygens is replaced by sulfur. Examples of phosphodiester and phosphorothioate internucleotide linkages are shown below:

The phosphorothioate bond substitutes a sulphur atom for a non-bridging oxygen in the phosphate backbone of an oligonucleotide, rendering the internucleotide linkage resistant to nuclease degradation. Phosphorothioates can be introduced at either the 5′- or 3′-end of the oligo to inhibit exonuclease degradation. In antisense oligonucleotides, phosphorothioates are also introduced internally to limit attack by endonucleases. The synthesis of phosphorothioate containing oligonucleotides is described, for example in Verma S. and Eckstein, F. (1998). MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users. Annu. Rev. Biochem. 1998. 67:99-134 and Curr Protoc Nucleic Acid Chem. 2009 March; Chapter 4: Unit 4.34. DNA oligonucleotides containing stereodefined phosphorothioate linkages in selected positions. Nawrot B, Rebowska B.
As mentioned above the sulfurisation of the internucleotide bond reduces the action of endo- and exonucleases2 including 5′→3′ and 3′→5′ DNA POL 1 exonuclease, nucleases S1 and P1, RNases, serum nucleases and snake venom phosphodiesterase. The nuclease resistant attribute of the S-oligo in conjunction with high fidelity PCR employing the use of exo+DNA polymerases has been demonstrated see, for example, Nucl. Acids Res. (2003) 31 (3): e7. doi: 10.1093/nar/gng007. Taq DNA polymerase possesses no 3′→5′ Exonuclease (Kenneth R. Tindall, Thomas A. Kunkel, Biochemistry, 1988, 27 (16), p 6008-6013). Enhanced discrimination of single nucleotide polymorphisms by phosphorothioate modification in the presence of a proof-reading polymerase has also been reported. Phosphorothioation increases specificity, reducing incidences of primer-dimer interactions, however it is reported that 3′ nuclease functionality is required for the improvement to PCR and it has been demonstrated that in conjunction with Allele Specific PCR the use of S-oligos offer no benefit when used in conjunction with Taq DNA polymerase see, for example, Zhang, J. and Li, K. (2003) Single-Base Discrimination Mediated by Proofreading 3′ Phosphorothioate-Modified Primers. Molecular Biotechnology 25, 223-227.
There is a need for easy-to-synthesise, low cost and reliable specific detection systems for use in the detection of primer extension products, e.g. in homogeneous PCR assays, which address the problems encountered with existing detection systems for PCR. Contrary to conventional scientific knowledge the present invention is based on the finding that S-oligos can be used successfully, and result in improvements, in nucleic acid detection assay systems.